Your Best Data: Teaming QIAGEN Chemistry & Bioinformatics...

Sample & Assay Technologies

Your Best Data: Teaming QIAGEN Chemistry & Bioinformatics to Drive

Samples to Insight

Aysel Heckel Director Clinical Solutions Sales

Dr. Anne Arens

Field Application Scientist

Course on Variant Detection and Interpretation in a Diagnostic Context

RS Erasmus MC Course 2014

Sample & Assay Technologies Sample to Insight

SSample Prep

AssayData

Sequence-Level

Statistics

Biology of Interest (Genes, Variants,

etc)

Comparative Statistical Analysis

Biological Interpretation

Upstream Analysis “primary”

Streamline Integration and Workflow Embedding

“secondary” “tertiary”

Better Results With Any Platform

Erasmus MC Course 2014

Sample & Assay Technologies Universal NGS workflow: from sample to insight

Overcome NGS challenges

Sample Isolation

Targeted Enrichment

Library Construction

NGS Run

Data Analysis

Interpretation

Sample Insight

Library QC

Sample QC

Variant Confirmation

Turnaround time, and limited

amounts of DNA

Library yield Platform specific

Amplification and

Sequencing

Data

processing & Variant calling

Isolation of high-quality DNA

samples

Quantifying amplifiable (not

bulk) amounts of DNA

Clinical & Biological

interpretation of data



Integrated solutions to address NGS challenges

Sample Isolation

Targeted Enrichment


NGS Run

Data Analysis

Interpretation

Sample Insight

Library QC

Sample QC


GeneRead DNAseq Targeted Panels V2

GeneRead DNAseq PCR Kit V2

GeneRead DNA Library Core & Amp Kits

GeneRead Adapter 12-plex GeneRead Size selection Kit GeneRead DNASeq Library Quant Array

Platform specific Amplification and Sequencing

CLC Cancer Research Workbench

GeneRead DNAseq V2 Data analysis

GeneRead DNA FFPE kit

GeneRead DNA QuantiMIZE kit

Ingenuity Variant

Analysis

  Ingenuity Clinical

Decision Support


Sample & Assay Technologies GeneRead DNA FFPE Kit

  The use of DNA isolated from FFPE tissues remains challenging

  Low DNA quality   Presence of base damage leading to artifactual single nucleotide

changes –  >50% of artifacts are C>T changes resulting from deamination

  Increase false positive rate

  GeneRead DNA FFPE Kit

  Enzymatically remove artifacts during DNA isolation step and provide more confidence in SNP calling

  Automate on QIAcube

5

Targeted high throughput sequencing in clinical cancer Settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissue, input amount and tumor heterogeneity

See also: Williams et al., 1999. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am J Pathol 155(5): 1467-1571 Yost et al., 2012. Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens. NAR 40(14): e108

Sample & Assay Technologies GeneRead DNA QuantiMIZE kits

Overcome the challenges of DNA quantification & qualification

Control and sample gDNA

Multi-copy qPCR assays +

Master Mix

Amount of input DNA and number of targeted

enrichment cycles

  Uses Innovative multi-copy qPCR assays to increase accuracy

  Two qPCR assays to query 40 genomic loci that are randomly distributed in the genome

  Quantifies only PCR-amplifiable DNA

  Provides guidance to rescue low-quality DNA samples

  Appropriate amount of input DNA

  Number of targeted enrichment cycles

  Generates libraries from low-quality FFPE samples

Need to quantify amplifiable “functional”, not bulk, DNA….. Why?



Overcoming the challenges of targeted enrichment prior to NGS

Sample Isolation

Targeted Enrichment


NGS Run

Data Analysis

Interpretation

Sample Insight

Library QC

Sample QC


GeneRead DNAseq Panel V2

GeneRead DNAseq PCR Kit V2

Gene 1 Gene 2 Multiplex PCR-based approach that enriches genes of interest to enable targeted DNA sequencing


Sample & Assay Technologies GeneRead DNAseq Targeted Panels V2

Minimal DNA input; Wet-bench validated panels

Actionable Mutations

Clinically-relevant

Tissue-specific

Comprehensive

Breast Cancer

Colorectal Cancer

Myeloid Neoplasms

Liver Cancer

Lung Cancer

Ovarian Cancer

Prostate Cancer

Gastric Cancer

Cardiomyopathy

Cancer

Carrier Testing

Cancer Predisposition

Only 40 ng DNA needed per panel (except Actionable Mutations panel which needs a total of 20 ng)

Largest collection of pre-designed panels suitable for a wide range of needs

RS

Mix-n-Match &

Custom


Sample & Assay Technologies Compatibility with major sequencing platforms

Same panel for different platforms

GeneRead® DNAseq Panels V2

HiSeq 2500 MiSeq

NextSeq

PGM Proton

RS

  Outstanding experimental performance   150 bp amplicons; FFPE-compatible   Enrichment in 3 hours   One library per sample   Customization tools to fit unique customer requirements

Booth


Sample & Assay Technologies GeneRead DNAseq Targeted Panels V2

Applications

Bone marrow samples

Single cells & circulating tumor cells

Biopsies & FFPE samples

Cell-free DNA

Liquid Biopsies

Abstract (Mon; 3384M): Single cell mutation detection with multiplex PCR-based targeted enrichment sequencing (Copies in back)

Presentation & Abstract (Sunday; 3353S) Evaluation of a multiplex PCR targeted enrichment approach for the detection of actionable mutations in FFPE samples via Next-Generation Sequencing Dr. Francine de Abreu Dartmouth-Hitchcock Medical Center (Copies in back)



To overcome NGS challenges

Sample Isolation

Targeted Enrichment


NGS Run

Data Analysis

Interpretation

Sample Insight

Library QC

Sample QC


Turnaround time, and limited

amounts of DNA

Library yield Platform specific

Amplification and

Sequencing

Data

processing & Variant calling

Isolation of high-quality DNA

samples

Quantifying amplifiable (not

total) amounts of DNA

Clinical & Biological

interpretation of data



Workflow


Interpretation Annotation Variant calling Assembly

Raw DNA sequence data

Sequencing

Report with valuable insights

Offering bioinformatics gold standards

QIAGEN has strong solutions for all worksteps

Strengthening leadership in offering universal bioinformatics solutions

Alignment to reference genome

Variant database & mutation content

Curated knowledge databases

Disease/Biological models

Reporting

CLC bio Ingenuity

Analysis & visualization



QIAGEN Bioinformatics Products for data analysis and interpretation of cancer and other human diseases


Sample & Assay Technologies Sample to Insight

Streamline Integration and Workflow Embedding

QIAGEN NGS solutions

Sample preparation

Data analysis Library construction

Target enrichment

Sample technologies Sequencing

•  CLC Cancer Research Workbench

•  Ingenuity Variant Analysis •  Ingenuity Clinical •  HGMD, PGMD, GenomeTrax •  Ingenuity Pathway Analysis


Sample & Assay Technologies CLC Cancer Research Workbench

  Easy to use   Human- and cancer-specific data can be updated

independently from the software   Comprehensive cancer end-to-end analysis workflows,

which can be customized   Ability to build own analysis workflows and share them

with collaborators, colleagues, and friends   Specific tools for cancer data analysis

  Sample genotyping   Primer removal after read mapping

  Sample comparison   Fast and easy analysis of Whole Genome, Whole

Exome, Targeted Amplicon and Whole Transcriptome Sequencing data

  New in October:   Integration with Ingenuity Variant Analysis for best in

class interpretation   Copy Number Detection tool from targeted data


Sample & Assay Technologies CLC Cancer Research Workbench

End-to-end analysis workflows and results

QC reports

Visualization



Analysis of QIAGEN GeneRead DNAseq Panel data

QIAGEN GeneRead DNAseq Panel Analysis Plugin

More info needed? www.clcbio.com/clc-plugin/qiagen-generead-panel-analysis

Identify and annotate SNVs, as well as small and large insertions and deletions in target amplicon sequencing data generated with QIAGEN’s GeneRead DNAseq Gene Panels Analysis of standard panels focused on a specific set of genes as well as customized panels to include genes tailored to specific research interests Free of charge!


Sample & Assay Technologies The Most Adopted NGS Interpretation Platform

Number of biological samples uploaded into Ingenuity Variant Analysis


Sample & Assay Technologies Most Comprehensive Human Disease Variant Content

Number of expert-curated human phenotype-associated variant findings in p

*658,179 Ingenuity and HGMD curated findings


Sample & Assay Technologies QIAGENs Bioinformatics Products

•  Secure web platform for interpreting called human genomes

•  Smart interface to flexibly annotate & compare genomes, to shortlist candidate variants, genes, & gene sets

•  Statistically robust methods for probands, matched tumors, kindreds, and big cohorts

•  Share your results with collaborators

•  HGMD now included

Ingenuity® Variant Analysis™ in a nutshell


Sample & Assay Technologies Variant Analysis and HGMD in One Subscription



Fastq-to-insight solution for cancer

Identify candidate (somatic) variants Interpret candidate variants

First Fastq-to-insight solution from QIAGEN

Release: End of October



Visualization of causal variants in Ingenuity Variant Analysis

Visualization of results

Quality of Usual Suspects

Frequency of the variant in healthy population.

Assessment (Pathogenic, etc) and Gain or loss of function

Identify variants based on a basic association test comparing case and control samples.

Causal Networks to identify biologically relevant variants.



Visualization and validation of causal variants from IVA in context of sequencing reads in CLC Cancer Research Workbench

Visualization of results



Plausible drivers for acinic cell carcinoma shortlisted by CLC Cancer Research Workbench and Ingenuity Variant Analysis

Case Story published in ClinicalOmics in 2014

plausible drivers

25255 likely acinar-

relevant 7

original

14


Sample & Assay Technologies Actionable Mutations Results – Variants

Sample ID Target mutation

MiSeq - Actionable Qiagen Panel

PGM - AmpliSeq Panel

Filtered Coverage

Variant Frequency Variant Frequency Mutation detected by

panel?

Mutation called by GeneRead

tool? Comments

1 EGFR del19 2497 46.80 46.70 Yes Yes

2 EGFR p.T790M - 3.00 5.00 Yes (3% as visualized

by IGV - see supporting material)

No

Mutation can be detected by thepanel; calling the mutation

requires the use of CLC Bio's algorithms for low frequency

mutations

3 KRAS G12V 2499 35.90 38.00 Yes Yes

4 BRAF V600E 2500 37.30 38.40 Yes Yes

5 IDH1 p.R132H 2498 43.80 45.30 Yes Yes

6 IDH2 R172K 1567 39.90 41.40 Yes Yes

7 BRAF V600E 2499 22.10 23.10 Yes Yes

8 NRAS Q61K 2499 45.30 48.80 Yes Yes

9 EGFR ins 2499 44.70 59.70 Yes Yes

10 NRAS Q61H 2500 10.10 11.00 Yes Yes BRAF V600E 2499 9.30 12.80 Yes Yes

Control 2

NRAS Q61K KIT D816V BRAF

V600E EGFR G719S KRAS G13D

2500 13.80 12.50 Yes Yes 2498 10.60 10.00 Yes Yes 2500 10.00 10.50 Yes Yes

22.20 24.50 Yes Yes 2492 15.10 15.00 Yes Yes


Sample & Assay Technologies Translational Research and Clinical Decision Support

Annotate, interpret, classify, report on clinically relevant variants

Sequencing Alignment Variant calling

Annotation, evidence synthesis

Biological interpretation / lab reporting

Upstream primary and secondary analysis Interpretation, classification, reporting

Complete analysis, interpretation and reporting workflow support

Called human variant

Basic, Translational and Clinical Research and Discovery

Variant Causality

NGS Workflow

Clinical Decision Support

Variant Interpretation & Reporting


Sample & Assay Technologies Evidence-based and configurable platform

Better and faster clinical assessments of observed variants

REPORTING engine for reporting to clinics and physicians – either

results export or template enabled

Pre-curated CLINCIAL EVIDENCE, DRUG LABELS, GUIDELINES,

TRIALS for variant classification and treatment selection

Rules-based decision support SOFTWARE that provides computed classifications based upon evidence

and lab defined rules

Customizable variant annotation, filtering and classification, reporting

WORKFLOW support


Sample & Assay Technologies Enabling scalable test interpretation and reporting

Automation of annotation, filtration, classification, reporting

Informatics / Software

Technician Clinical lab staff

Patient

Ordering physician Test

definition

Clinical sample

Raw test data

Clinical Geneticist

Test order

Variant Calls

Public Literature

Test details

Called variants

Patient/ clinical

data

Internal / LSDBs

Lab Director

Classification & Treatment

Selection

Internal Report

Lab sets up test product, selects classification rules and

reporting methods

Test order includes patient and relevant clinical information

Lab director reviews clinical assessment and signs out

report

Ingenuity Clinical is an evidence-based decision support system intended as an aid in the interpretation of variants observed in genomic sequencing data. The program evaluates genomic variants in the context of published biomedical literature, professional association guidelines, and publicly available databases and annotations. Based on this evaluation, the Ingenuity Software proposes a classification and bibliographic references to aid in the interpretation of observed variants. The software is NOT intended as a primary diagnostic tool by physicians or to be used as a substitute for professional healthcare advice.


Patholoigist /Oncologist

Sample & Assay Technologies Customizable Reporting


Your Best Data: Teaming QIAGEN Chemistry & Bioinformatics...

Documents

Transcript of Your Best Data: Teaming QIAGEN Chemistry & Bioinformatics...